PROJECT SUMMARY Recurrent urinary tract infections (rUTI) are among the most common bacterial infections, causing an estimated 268,000 cases in the US alone. Bacteria such as the uropathogenic Escherichia coli (UPEC) follow a multistep, pathogenic cycle in which intracellular bacterial communities (biofilms) are established within the urothelium during infection. The opportunistic pathogen Pseudomonas aeruginosa also forms highly resistant biofilms and infects chronically-catheterized patients. The inability of current prophylactic antibiotic regimens to access the long-lived intracellular bacterial reservoirs within the urothelium renders antibiotics inefficient while contributing to resistance. We propose a novel approach to treat rUTI in which targeted nanogels with high loading of multiple antibiotics can be delivered transurethrally to the bladder wall. We recently published results in which it was demonstrated that the cell penetrating peptide CGCKRK, when covalently conjugated on the surface of a biocompatible acrylic nanogel, can efficiently bind and penetrate the denuded/injured murine bladder wall in-vivo and successfully deliver a drug mimic within the murine urothelium. Based on the in vitro and in vivo preliminary data gathered, we have detailed a project plan in which nanogel networks that penetrate the urothelium to enable the sustained delivery of antibiotics to successfully treat rUTIs will be engineered. We will expand on our preliminary methods by 1) synthesizing nanogels with enhanced capacity to encapsulate and release therapeutically relevant doses of antibiotics (ciprofloxacin and gentamicin) in physiologically relevant timescales and tested in vitro and; 2) establish the conditions to successfully treat rUTI in an in-vivo murine infection model. Additional potential of the targeted bladder delivery systems described in this proposal is the ability to simultaneously deliver multiple therapeutics, such as a single antibiotic, an antibiotic cocktail or a biofilm dispersing molecule. The development of a bladder delivery system described here could also be used to advance treatments of other urinary bladder pathologies.